Method for detecting disease-associated mutations

ABSTRACT

A method is described for diagnosing individuals as having hypertrophic cardiomyopathy, e.g. familial or sporadic hypertrophic cardiomyopathy. The method provides a useful diagnostic tool which becomes particularly important when testing asymptomatic individuals suspected of having the disease. Symptomatic individuals have a much better chance of being diagnosed properly by a physician. Asymptomatic individuals from families having a history of familial hypertrophic cardiomyopathy may be selectively screened using the method of this invention allowing for a diagnosis prior to the appearance of any symptoms. Individuals having the mutation responsible for the disease may be counseled to take steps which hopefully would prolong their life, i.e. avoid rigorous exercise. The methodology used in the above method also has broad applicability and may be used to detect other disease-associated mutations in DNA obtained from subject being tested for other disease-associated mutations.

RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 10/788,779, filed Feb. 27, 2004, pending, which isa continuation of U.S. application Ser. No. 08/469, 172, filed Jun. 6,1995, pending, which, in turn, is a continuation application of U.S.application Ser. No. 07/989,160, granted as U.S. Pat. No. 5,429,923,filed on Dec. 11, 1992. The contents of all of the aforementionedapplications are hereby incorporated by reference.

BACKGROUND

The use of an individual's genetic information in the diagnosis of adisease is becoming more prevalent. Many diseases are caused by a defectin a single gene of an individual. All known autosomal dominant,autosomal recessive and x-linked disorders are believed to be caused bya defect in a single gene (Antonarakis, New England Journal of Medicine,Vol. 320, No. 3:153-63 (1981)). Genes responsible for some diseases ordisorders have been cloned and characterized. The defect in the gene maybe a gross gene alteration, a small gene alteration or even a pointmutation. Examples of some diseases caused by a mutation in a geneinclude Gaucher's disease, hemophilia A and B, Duchenne's musculardystrophy, sickle cell anemia, Tay-Sachs disease, phenylketonoria andcystic fibrosis.

Familial hypertrophic cardiomyopathy (hereinafter FHC) has been linkedto mutations in the β cardiac myosin heavy-chain gene (Tanigawa et al.,Cell 62:991-998 (1990)); Geisterfer-Lowrance et al., Cell 61:999-1006(1990)). Tanigawa et al. studied a single family (Family B) andhypothesized that the FHC in this family was due to a mutation thatresults in the formation of an α/β cardiac myosin heavy-chain hybridgene. Geisterfer-Lowrance et al. also studied a single family andhypothesized that a missense mutation in the β cardiac myosinheavy-chain gene caused FHC in the family studied.

FHC is a well characterized autosomal dominant disorder or disease. Itis autosomal dominant in that fifty percent of the children of affectedparents eventually become afflicted with the disease. FHC ischaracterized by unexplained myocardial hypertrophy. The clinicalsymptoms of individuals having FHC are variable and some individuals donot have any symptoms. The symptoms of FHC include dypsnea, angina,ischemia. Pathological findings of the disease include increasedmyocardial mass with myocyte and myofibrillar disarray.

Presently, the diagnosis of individuals having FHC relies on thepresence of typical clinical symptoms and the demonstration ofunexplained ventricular hypertrophy. Sudden, unexpected death is themost serious consequence of FHC. Sudden death occurs in both symptomaticand asymptomatic individuals and FHC has an annual mortality ofapproximately four percent from sudden death.

SUMMARY OF THE INVENTION

The present invention provides a method for diagnosing individuals ashaving hypertrophic cardiomyopathy (hereinafter HC), e.g. familial orsporadic hypertrophic cardiomyopathy (hereinafter FHC or SHC). Themethod provides a useful diagnostic tool which becomes particularlyimportant when screening asymptomatic individuals suspected of havingthe disease. Symptomatic individuals have a much better chance of beingdiagnosed properly by a physician. Asymptomatic individuals fromfamilies having a history of FHC may be selectively screened using themethod of this invention allowing for a diagnosis prior to theappearance of any symptoms. Individuals having the mutation responsiblefor FHC may be counseled to take steps which hopefully would prolongtheir life, i.e. avoid rigorous exercise.

A method involving both an amplification and detecting step fordetecting mutations associated with hypertrophic cardiomyopathy has notbeen previously described. The present method for detecting the presenceor absence of a mutation associated with hypertrophic cardiomyopathyinvolves amplifying β cardiac myosin heavy-chain DNA forming anamplified product and detecting the presence or absence of a mutationassociated with hypertrophic cardiomyopathy in the amplified product.

The present invention further pertains to a method for diagnosingfamilial hypertrophic cardiomyopathy. Prior to the present invention,there were no extensive studies involving a large number of familieswhich established that this disease or disorder was caused by pointmutations in the β cardiac myosin heavy-chain gene when the causativemutation is located within this gene. The process of diagnosing adisease caused by a point mutation is considerably more complex ifmultiple genes and multiple point mutations are responsible for theparticular disease. FHC falls into this complex category because it isdue to defects in the β cardiac myosin heavy-chain gene in approximately50% of the families and unrelated families have differentdisease-causing point mutations. The present invention is based, atleast in part, on the discovery that FHC is caused by point mutationswhen the mutation involves the β cardiac myosin heavy-chain gene andeven further that different unrelated families have differentdisease-causing point mutations. The large size of the gene makesidentifying disease-causing mutations laborious. The present inventionprovides a relatively rapid and easy method for accomplishing thisdifficult task.

The method for diagnosing FHC includes obtaining a sample of β cardiacmyosin heavy-chain DNA derived from the subject being screened for FHCand diagnosing the subject for FHC by detecting the presence or absenceof a FHC-causing mutation in the β cardiac myosin heavy-chain DNA as anindication of the disease. The β cardiac myosin heavy-chain DNA may becDNA reverse transcribed from RNA obtained from the subject's bloodlymphocytes.

The present invention also provides a non-invasive method for diagnosingHC that exploits the ectopic expression of this gene in nucleated bloodcells, e.g., peripheral-blood mononuclear cells, allowing for access toβ cardiac myosin heavy-chain transcripts from peripheral blood. Accessto β cardiac myosin heavy-chain transcripts in peripheral blood permitsefficient amplification of coding sequences which can be analyzed forsmall deletions, alternative splicing or point mutations with RNaseprotection assays. The non-invasive method for diagnosing HC involvesobtaining a blood sample from a subject being screened for HC andisolating β cardiac myosin heavy-chain RNA from the blood sample. Thesubject is diagnosed for HC by detecting the presence or absence of anHC-associated mutation in the RNA as an indication of the subject havingthe disease. Mutations in the RNA may be detected by reversetranscribing the RNA into cDNA and subsequently detecting HC-associatedmutations in the cDNA.

The present invention further provides a method that allows thedetection of disease-causing mutations in a DNA sequence associated witha disease. Screening for a mutation in a person at risk for a particulardisease can be accomplished rapidly and relatively easily through thepresently described-method. The method of this invention may be used todetect mutations responsible for diseases or disorders such ashypertrophic cardiomyopathy, e.g. familial or sporadic, cystic fibrosis,Gaucher's disease, hemophilia A and B, Duchenne's muscular dystrophy,sickle cell anemia, Tay-Sachs disease, and phenylketonuria.

The method for detecting the presence or absence of a disease-associatedmutation in a DNA sequence involves amplifying a DNA sequence suspectedof containing a disease-associated mutation forming an amplifiedproduct, combining the amplified product with an RNA probe completelyhybridizable to a normal DNA sequence associated with the diseaseforming a hybrid double strand having an RNA and DNA strand.Subsequently, the hybrid double strand is contacted with an agentcapable of digesting an unhybridized portion of the RNA strand and thepresence or absence of an unhybridized portion of the RNA strand isdetected as an indication of the presence or absence of adisease-associated mutation in the corresponding portion of the DNAstrand. The method of this invention may be used to detect mutationswhich are reflected in the RNA. The method may be used to detectmutations of a size which is less than or equal to the amplified pieceof DNA defined by the primers. Preferably, the mutation is less thanabout 500 bp, more preferably less than about 100 bp, even morepreferably less than about 10 bp, and most preferably a point mutation,i.e. a change in a single nucleotide.

Other aspects of this invention pertain to kits including containersholding reagents used in the above-described methods and a method fordetermining the estimated life expectancy of a person having FHC usingthe above-described methods. The components of the kit also are part ofthis invention. These aspects are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nested polymerase chain reaction (hereinafter PCR)used to amplify a β cardiac myosin heavy-chain complementary DNA (cDNA)with nucleotide residues indicated by numbers. Reverse transcriptase wasused to obtain the cDNA from mRNA extracted from peripheral-bloodmononuclear cells or cell lines transformed by Epstein-Barr virus. ThecDNA was used as a template for the initial PCR with primers A and B orC and D. The resulting products were diluted 1:1000, and PCR wasrepeated with internal primers A′ and B′ or C′ and D′. The positions ofthe missense mutations found in the previously described Family A(residue 1294) and in presently described Family QQ (residue 832) areindicated. The cDNA fragment used as the template for the riboprobe usedin RNase protection assays (shown in FIG. 2) is also indicated.

FIG. 1B is a photograph showing the normal and mutant β cardiac myosinheavy-chain transcripts after PCR amplification. Products are shown on athree percent NuSieve and one percent agarose gel stained with ethidiumbromide. Lane 1 (Uncut) contains a PCR product (275 bp) derived fromnormal peripheral-blood mononuclear cells with primers C and D and C′and D′. Lanes 2 through 5 contain PCR products derived from unaffectedor affected members of Family A and digested with the restriction enzymeDdel. The 180 bp fragment is present only in products from affectedfamily members.

FIG. 2A is a schematic showing the identification of mutations in the βcardiac myosin heavy-chain gene using an RNA protection assay. A³²P-labeled RNA probe (shown in FIG. 1A) was transcribed in vitro from afragment of wild-type B cardiac myosin heavy-chain cDNA. Amplifiedproducts generated by nested PCR were hybridized to the RNA probe, andthe resulting RNA-DNA hybrid was digested with RNase A. Overhanging endsof the probe and mismatched bases (hatched areas) were digested by RNaseA. Digestion products were analyzed by denaturing acrylamide-gelelectrophoresis. The results of electrophoresis is shown in the box.Samples from homozygous, unaffected persons (U/U) contained amplifiedfragments homologous to the β cardiac myosin heavy-chain RNA probe(single bold band). Samples from heterozygous, affected persons (U/A)contained these fragments (upper band) and new fragments (two lowerbands) resulting from internal cleavage at the site of the mismatchbetween the wild-type β cardiac myosin heavy-chain probe and productsamplified from the mutant gene.

FIG. 2B is a photograph showing the results of the RNA protection assayin two affected probands and in members of Family QQ. Products of nestedPCR obtained with primers A and B and A′ and B′ (shown in FIG. 1A) wereanalyzed in an RNase protection assay with the riboprobe shown in FIG.1A. Samples from two unrelated, affected probands are shown in lanes 1and 2. Lanes 3 through 14 contain samples from members of Family QQ,identified according to the pedigree shown in FIG. 3 and disease status.The RNase protection assay identified a novel fragment (arrow) in familymember I-1 that is present only in affected members of Family QQ. ThePCR controls produced no fragments visible on ethidium bromide stainingof agarose gel and did not protect the β cardiac myosin heavy-chain RNAprobe.

FIG. 3A is a schematic showing family pedigree of Family QQ. Male familymembers are denoted by squares, female members by circles, deceasedmembers by a slash, affected members by solid symbols, unaffectedmembers by open symbols, and members whose disease status undeterminedby stippled symbols. The disease status was based on clinical analysis.Numbered family members were available for clinical and geneticevaluation.

FIG. 3B shows the results of the genetic analysis of generation III.Genomic DNA was extracted from two independent blood samples obtainedfrom members of Family QQ and amplified by PCR with primers B9.1F andB9.1R. Products were digested with the restriction enzyme EcoRl andfractionated according to size on a three percent NuSieve and onepercent agarose gel stained with ethidium bromide. Samples from personswith the normal sequence contained two fragments consisting of 79 and 45bp. Samples from persons with the mutant sequence contained thesefragments and the full-length 124 bp fragment because the QQ mutationabolishes the internal EcoRl site. Analysis of family member III-14 wasperformed independently.

FIG. 4A depicts the detection of the mutations using the RNaseprotection assay described in Example 2. The figure shows the locationwithin normal human β cardiac myosin heavy-chain (MHC) RNA (shaded bar)of the sequences of a proband's DNA and the riboprobe templates used inthe RNase protection assay. The segments of DNA used for protection arecDNA segments 1 through 5, derived from nucleated blood cells, e.g.peripheral-blood mononuclear cell; RNA (heavy lines represent theinitial PCR production, and light lines the products of a secondamplification with an inner primer pair), and exons from genomic DNA(see the Examples set forth below). The eight templates used asriboprobes, numbered according to nucleotide residue, are shown in thebottom half of the panel. Segment 3421-3811 was amplified from exon 27,not mRNA as described below.

FIG. 4B shows the results of an RNase protection assay and six probands.Exon 16 was amplified from the genomic DNA of probands from Families DD,LL and L and three unrelated probands (P). The amplified DNA protects apredominant RNA fragment 310 bases long that is present in every person.Novel pairs of protected RNA fragments present in Families LL and DD(170 and 140 bases, respectively) and Family L (235 and 75 bases) resultfrom cleavage of the riboprobe, indicating a mismatch between thesequences of the DNA of these probands and the sequence of normal myosinheavy-chain DNA.

FIG. 5 depicts the location and identity of missense mutations infamilies with familial hypertrophic cardiomyopathy. A schematic diagramof the normal cardiac myosin heavy-chain gene is shown in the center (5′to 3′) and the location of the missense mutations is shown according toexon. The amino acid substitutions predicted by each mutation are shownin the top of each box, and the families with these mutations aredesignated by letters. Sequences that encode the initiation oftranscription (ATG), ATPase activity (ATP), actin binding (Actin I andActin II), myosin light-chain binding (MLC), and the hinge function(Hinge) are indicated. The head and rod regions of the encodedpolypeptide are shown at the bottom the figure.

FIG. 6 depicts Kaplan-Meier product-limit curves for the survival offamily members according to mutation. The curves are shown for familieswith each of four mutations. The curve for Arg453Cys refers to affectedmembers in families with and without the hybrid gene.

DETAILED DESCRIPTION

The present invention pertains to a method for detecting the presence orabsence of a mutation associated with hypertrophic cardiomyopathy. Themethod involves amplifying β cardiac myosin heavy-chain DNA to form anamplified product and detecting the presence or absence of a mutationassociated with HC in the amplified product.

The term mutation for purposes of this invention is intended to includemutations associated with the respective diseases being discussed, e.g.hypertrophic cardiomyopathy. The mutation may be a gross alteration inthe RNA or DNA, small alteration in the RNA and DNA, or even a pointmutation in the RNA or DNA. The mutation further may be a mutation ofthe DNA which changes the amino acid encoded by that portion of the DNAstrand, e.g. a missense mutation, or a mutation which does not changethe encoded amino acid.

HC is a well characterized disorder or disease as described above. Thisterm is intended to include both FHC or SHC. FHC is inherited throughoutfamilies and SHC occurs sporadically without a traceable hereditarypath. For example, a subject having HC clinical symptoms may bediagnosed as being SHC if both of the subject's parents are actuallydiagnosed and determined to be healthy yet the subject has HC. Evenfurther, if an afflicted subject's parents are not available fordiagnosis and the afflicted subject has no other known family memberswith HC, then the subject probably would be diagnosed as having SHC.

The term amplification for purposes of this invention is intended toinclude any method or technique capable of amplifying the respective DNA(including culturing) or RNA being discussed. The preferredamplification technique is the polymerase chain reaction (PCR) which isan art recognized technique and most preferably the amplification isconducted using a nested PCR technique as described in the examplesbelow.

The term β cardiac myosin heavy-chain DNA for purposes of this inventionincludes both genomic β cardiac myosin heavy-chain DNA and β cardiacmyosin heavy-chain cDNA. The preferred β cardiac myosin heavy-chain DNAis cDNA reverse transcribed from RNA obtained from a subject beingscreened for the respective disorder or disease, e.g. SHC or FHC. TheRNA may be obtained from cardiac or skeletal tissue or from nucleatedblood cells as described below.

The detection of the presence or absence of a mutation associated withhypertrophic cardiomyopathy in the amplified product may be conductedusing any method capable of detecting such mutations. Examples ofconventional methods used to detect mutations in DNA sequences includedirect sequencing methods (Maxim and Gilbert, PNAS USA 74:560-564(1977); Sanger et al., PNAS USA 74:5463-5467 (1977)), homoduplexmethods, heteroduplex methods, the single-stranded confirmation ofpolymorphisms (SSCP analysis) technique, and chemical methods. It shouldbe understood that these methods are being provided merely to illustrateuseful methods and one of ordinary skill in the art would appreciateother methods which would be useful in the present invention. Thepreferred detection method of the present invention is a heteroduplexmethod, particularly a protection assay which is similar to the RNaseprotection assay described by Myers et al. (Science, Vol 230, No.3:1242-46 (1985)), the contents of which is expressly incorporated byreference.

A protection assay may be used to detect the presence or absence of theHC-causing mutation by combining amplified β cardiac myosin heavy-chainDNA with an RNA probe under hybridization conditions forming a hybriddouble strand. The RNA probe is selected to be completely hybridizableto normal β cardiac myosin heavy-chain DNA, i.e. DNA withoutdisease-causing mutatons. The hybridization conditions are the same orsimilar to those described by Myers et al., cited supra. For example,the hybridization may include the addition of the RNA probe to asolution containing the DNA, e.g. a hybridization buffer, at appropriateconditions, e.g. 90° C. for ten minutes. Subsequently, this mixture maybe incubated for a longer period of time, e.g. at 45° C. for thirtyminutes.

The term “completely hybridizable” for purposes of this invention isintended to include RNA probes capable of hybridizing at each nucleotideof a complementary normal DNA sequence. This characteristic of the RNAprobe allows for the detection of an unhybridized portion at amismatched or mutant nucleotide(s).

The hybrid double strand, i.e. the RNA:DNA double strand, hasunhybridized portions of RNA at locations or portions corresponding to amutation in the normal DNA strand, e.g. an HC-associated mutation. Thehybrid double strand is contacted with an agent capable of digesting anunhybridized portion(s) of the RNA strand, e.g. an RNase. The presenceor absence of any unhybridized portions are then detected by analyzingthe resulting RNA products. The RNA products may be analyzed byelectrophoresis in a denaturing gel. Two new RNA fragments will bedetected if the sample DNA contained a point mutation resulting in anunhybridized portion recognizable by the RNase. The total size of thesefragments should equal the size of the single RNA fragment resuting fromthe normal DNA. The mutation(s) can be localized relative to the ends ofthe RNA probe by determining the size of the new RNA products. Thesequence of the mutation may be determined by looking at the localizedportion of corresponding DNA.

The agent capable of digesting an unhybridized portion of the RNA strandmay be any agent capable of digesting unprotected ribonucleotides in thehybrid strands. Examples of such agents include ribonucleases,particularly RNase A.

As set forth above, the method of this invention can detect the presenceor absence of the mutation associated with the respective disease oreven further, the position within the gene or sequence of the mutation.The sequence or position may be determined by observing fragmentsresulting from mutations and comparing the fragments to a known templatederived from the riboprobe which is representative of normal DNA.

The present method further pertains to a method for diagnosing FHC byobtaining a sample of β cardiac myosin heavy-chain DNA derived from asubject being screened for FHC. The subject is diagnosed as having FHCby detecting the presence or absence of an FHC-causing point mutation inthe β cardiac myosin heavy-chain DNA as an indication of the disease.

The term subject for purposes of this invention is intended to includesubjects capable of being afflicted with HC. The preferred subjects arehumans.

The present invention is based, at least in part, on the discovery thatFHC is caused by point mutations in the β cardiac myosin heavy-chaingene. Prior to the present invention, there were no extensive studiesinvolving a large number of families which established that thisparticular disease or disorder was caused by point mutations in the βcardiac myosin heavy-chain gene. Geisterfer-Lowrance et al. (Cell62:999-106 (1990)) described a point mutation in exon 13 of the βcardiac myosin heavy-chain gene which was present in all individualsaffected with FHC from a large family. Tanigawa et al. (Cell 62:991-998(1990)) determined that an α/β cardiac myosin heavy-chain hybrid gene iscoinherited with FHC in a different family (Family B). In view of bothof these findings, it was not clear until the present invention that FHCis caused by a point mutation and not a hybrid gene.

The present invention further pertains to a non-invasive method fordiagnosing hypertrophic cardiomyopathy. The method involves obtaining ablood sample from a subject being screened for hypertrophiccardiomyopathy, isolating β cardiac myosin heavy-chain RNA from theblood sample, and diagnosing the subject for hypertrophic cardiomyopathyby detecting the presence or absence of a hypertrophiccardiomyopathy-associated mutation in the RNA as an indication of thedisease.

The RNA may be isolated from nucleated blood cells. Nucleated bloodcells include lymphocytes, e.g. T and B cells, monocytes, andpolymorphonuclear leucocytes. The RNA may be isolated using conventionaltechniques such as isolation from tissue culture cells, guantidiniummethods and the phenol/SDS method. See Ausebel et al. (Current Protocolsin Molecular Biology (1991), Chapter 4, Sections 4.1-4.3), the contentsof which are expressly incorporated by reference.

The present invention is based, at least in part on the discovery, thatnormal and mutant β cardiac myosin heavy-chain RNA is present innucleated blood cells, e.g. lymphocytes, a phenomenon called ectopictranscription. Access to RNA provides a more efficient method ofscreening for disease-causing mutations because intron sequences havebeen excised from these transcripts. This is further advantageousbecause cardiac myosin heavy-chain RNA is abundant in the heart andslow-twitch skeletal muscle but its expression in other tissues isextremely low (Mahdavi et al., Nature 297:659-64 (1982); Lomprei et al.,J. Biol Chem 259:6437-46 (1984); and Lichter et al., Eur J Biochem160:419-26 (1986)). An invasive method would be required to obtain RNAfrom the aforementioned muscles whereas the present invention is anon-invasive method in that the mRNA is easily obtained from a bloodsample.

The present invention further pertains to a method for detecting thepresence or absence of a disease-associated mutation in a DNA sequence.This method is carried out by amplifying a DNA sequence suspected ofcontaining a disease-associated mutation forming an amplified product,and combining the amplified product with an RNA probe completelyhybridizable to a normal DNA sequence associated with the diseaseforming a hybrid double strand having an RNA and DNA strand. The hybriddouble strand has unhybridized portions of the RNA strand at anyportions corresponding to a disease associated mutation in the DNAstrand. The presence or absence of an unhybridized portion of the RNAstrand is detected as an indication of the presence or absence of adisease associated mutation in the corresponding portion of the DNAstrand. The presence or absence of an unhybridized portion of the RNAstrand may be detected by contacting the hybrid double strand with anagent capable of digesting an unhybridized portion of the RNA strand,denaturing the hybrid double strand, separating the RNA fragments bysize, and detecting the presence or absence of fragments of RNAresulting from portions of an RNA strand being digested by the agent.The method further may include the sequencing of a portion of DNAcorresponding to an unhybridized portion of the RNA strand to identifythe sequence of a disease-associated mutation. More than one mutationalso may be detected using the method of the present invention.

Many diseases have already been established as being associated with amutation in the DNA sequence, e.g. a particular gene. A diseaseassociated mutation for purposes of this invention includes a mutationlinked to or believed to be at least part of the causative factor forthe disease. Some diseases associated with mutations have been describedin Antonarakis, cited supra, the contents of which is expresslyincorporated by reference. Antonarakis describes an expansive list ofdisorders or diseases, the gene associated with such diseases, thelocation on a particular chromosome of the gene, and the types ofmutations. Some of the diseases associated with mutations and particulargenes are as follows: (each disease is followed by the respective gene)Gaucher's disease (glycocerebrosidase), Factor XIII deficiency (FactorXIII), diabetes mellitus due to abnormal insulins (insulin), sickle cellanemia, β-thalassemia (β-globin), McArdle's disease (muscle glycogenphosphorylase), phenylketonuria(phenylalanine hydroxylase), Tay-Sachsdisease (α₁-hexosaminidase), α-thalassemia (α-globin), Duchenne'smuscular dystrophy (gene for Duchenne's muscular dystrophy), hemophiliaB (Factor IX), and hemophilia A (Factor VIII). It should be understoodthat the method of this invention may also be used for detecting amutation associated with HC as described above.

The present invention also pertains to a method for determining theestimated life expectancy of a person having FHC. The method involvesobtaining 8 cardiac myosin DNA derived from a subject having FHC anddetecting a FHC-causing point mutation. The point mutation subsequentlyis classified as a particular type and the life expectancy of thesubject is estimated using a Kaplan-Meier curve for the classified typeof mutation. This aspect of the invention is described in more detailbelow.

The present invention also pertains to kits useful for diagnosing HC.The kit contains a first container such as a vial holding an RNA probeand a second container holding primers. The RNA probe is completelyhybridizable to β cardiac myosin heavy-chain DNA and the primers areuseful for amplifying β cardiac myosin heavy-chain DNA. The kit furthermay contain an RNA digesting agent or instructions for using thecomponents of the kit to detect the presence or absence of HC-associatedpoint mutation in amplified β cardiac myosin heavy-chain DNA. The RNAprobe and primers also are intended to be part of this invention.

The following examples are being provided to further illustrate theabove-described invention and should in no way be construed as beingfurther limiting to the present invention. The entire contents of all ofthe references mentioned in the below examples are expresslyincorporated by reference. The entire contents of Rozensweig et al. (NewEngland Journal of Medicine 325:1753-60 (Dec. 19, 1991)) and Watkins etal. (New England Journal of Medicine 326:1108-1114 (Apr. 23, 1992)) alsoare expressly incorporated by reference.

EXAMPLE 1 The Detection of a Missense Mutation in the β Cardiac MyosinHeavy-Chain Gene in Members from Family A and Family OO GeneralMethodology

Cell Lines and DNA and RNA Extraction

Blood was drawn from members of Family A and normal control subjects.The blood samples were used to prepare DNA from red-cell pellets(Gross-Bellard et al., Eur. J. Biochem. 36:32-8 (1973)) and to establishlymphoblastoid cell lines (Holcombe et al., Genomics 1:287-91 (1987)).RNA was prepared from fresh peripheral-blood mononuclear cells orEpstein-Barr virus-transformed cell lines by acid guanidiniumthiocyanate-phenol-chloroform extraction (Chomczynski et al., Anal.Biochem. 162:156-9 (1987)).

PCR and Restriction Enzyme and Sequence Analysis

Nested PCR (Sarkar et al., Science 244:331-4 (1989)) was used to amplifyβ cardiac myosin heavy-chain RNA from fresh peripheral-blood mononuclearcells and cell lines transformed by Epstein-Barr virus (see FIG. 1A).One to 2 μg of total RNA was reverse-transcribed with Moloney murineleukemia virus reverse transcriptase (Bethesda Research Laboratories)with 0.5 μg of the antisense primer from the outer primer pair. Thefirst round of amplification was then performed by the addition of 0.5μg of the outer sense primer (FIG. 1A, A or C) and 0.2 mmol of eachdeoxynucleoside triphosphate (Pharmacia) in a volume of 100 μl (finaldilution, 1:1000) containing 10 mmol of TRIS-hydrochloric acid (pH 8.3),50 mmol of potassium chloride, 1.5 mmol of magnesium chloride, and 0.01percent (wt/vol) gelatin. Forty cycles were carried out in athermocycler (Perkin-Elmer Cetus) under the following conditions: 0.5minute of denaturation at 94° C., one minute of primer annealing at 55°C., and two minutes of primer extension at 72° C. PCR products were thendiluted 1:100 and 10 μl was used as the template for the reaction in avolume of 100 μl of PCR buffer for Amplitaq (sold by Perkin-Elmer)(final dilution, 1:1000), in which the inner primer pair (FIGS. 1A, A′and B′ or C′ and D′) was used for an additional forty cycles. After thesecond reaction, 10 μl of the PCR product was electrophoresed on a twopercent agarose gel to confirm amplification. To avoid contamination ofthe PCR products, positive displacement or filtered pipette systems wereused and a number of negative controls were run with each amplification.Restriction analysis of these products was performed according topreviously described techniques (Ausebel et al. Current Protocols inMolecular Biology, 1989; Sanbrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbors, 1989). The PCR products weresequenced by performing an additional round of asymmetric amplification(Ausebel et al. cited supra (1989)) followed by direct sequenceanalysis, as previously described for single-stranded products (Sangeret al. PNAS USA 74:5463-7 (1977)). Genomic DNA was amplified for thirtyfive cycles with primers B9.1F and B9.1R, including denaturation for 0.5minutes at 94° C., primer annealing for one minute at 55° C. and primerextension for one minute at 72° C. The sequences of the PCR primers wereas follows: A, 5′CAAGGATCGCTACGGCTCCTGGAT3′; B,5′GCGGATCCAGGTAGGCAGACTTGTCAGCCT3′; A′, 5′ATGCCAACCCTGCTCTGGAGGCCT3′;B′, 5′CTTCATGTTTCCAAAGTGCATGAT3′; C, 5′CTGGGCTTCACTTCAGAGGAGAAAA3′; D,5′GCGGTACCCCAGCAGCCCGGCCTTGAAGAA3′; C′,5′GGGAATTCGCGGAGCCAGACGGCACTGAAG3′; D, 5′CCCTCCTTCTTGTACTCCTCCTGCTC3′;B9.1F, 5′CAACTCATCACCACTCTCTTCCATC3′; and B9.1R,5′GCTGAGCCTAGCAGATTCATGGCAC3′.RNase Protection

RNase protection was performed as described by Myers et al. (Science230:1242-6 (1985)) with the use of volumes scaled down threefold (seeFIG. 2A). First, PCR-amplified product (4 ul) was hybridized to a³²P-labeled RNA probe (200,000 counts per minute) and the resultingRNA-DNA hybrid was then digested with RNase A (Sigma) and analyzed bydenaturing acrylamide-gel electrophoresis. RNase reactions were stoppedby the simultaneous addition of proteinase K and sodium dodecyl sulfateand 15 μl of the final product was added to 20 μl of loading buffer forelectrophoresis without phenol-chloroform extraction or ethanolprecipitation.

Clinical Evaluation

Family members were evaluated by physical examination, 12-leadelectrocardiography, Doppler ultrasonography, and two-dimensionalechocardiography, with left and right ventricular views (Maron et al.,Am J Cardiol 48:418-28 (1981); Shapiro et al., J Am Coll Cardiol2:437-44 (1983); McKenna et al., 11:351-8 (1988)). Electrocariogramswere interpreted according to standard criteria (Surawicz et al., Am JCardiol 41:130-45 (1978)). Echocardiographic measurements of wallthickness and cavity dimensions and the presence or absence of systolicanterior motion of the mitral valve were determined according toestablished protocols (Maron et al., cited supra; Shapiro et al., citedsupra; McKenna et al., cited supra; Wigle et al. Prog Cardiovasc Dis28:1-83 (1985)). The diagnosis of familial hypertrophic cardiomyopathywas based on the demonstration of unexplained ventricular hypertrophy.Clinical diagnoses were made by two experienced clinicians who had noknowledge of the genotypic results. None of the family members evaluatedhad a history of systemic hypertension or a blood pressure higher than140/90 mm Hg at rest.

The above-described general methodology was used in the example setforth below on samples obtained from members of Family A and controlsubjects.

Determination of Whether the β Cardiac Myosin Heavy-Chain Gene isEctopically Expressed in Blood Mononuclear Cells

It was decided to determine whether there was ectopic expression of theβ cardiac myosin heavy-chain gene in blood mononuclear cells. A strategyof nested PCR amplification was used to detect extremely low levels of βcardiac myosin heavy chain mRNA as shown in FIG. 1A. Reversetranscriptase was used to obtain the cDNA from RNA extracted fromperipheral blood mononuclear cells or cells transformed by Epstein-Barrvirus. The cDNA was used as a template in the initial round of PCR.After the first round of amplification with primers C and D (or A andB), no specific product was visible on ethidium bromide staining. Asecond round of PCR was then performed with internal primers C′ and D′(or A′ and B′) after a 1000-fold dilution of the initial products.Sequential amplification yielded a product of 275 base pairs (see FIG.1B, line 1), which is the size predicted for the β cardiac myosinheavy-chain sequence. Partial nucleotide-sequence analysis was performedon several PCR-generated fragments to demonstrate that the productsobtained were derived specifically from the β cardiac myosin heavy-chaingene. The sequence was identical to that previously published byJaenicke et al. (Genomics 8:194-206 (1990)).

Determination Whether Mutated as Well as Normal Transcripts of the βCardiac Myosin Heavy-Chain Gene are Detectable in Peripheral BloodMononuclear Cells

It was decided to determine whether mutated as well as normaltranscripts of the β cardiac myosin heavy-chain gene could be detectedin peripheral-blood mononuclear cells. Samples obtained from a familywith familial hypertrophic cardiomyopathy (Family A) were analyzed.Affected members of this family previously were shown to have a missensemutation in exon 13 of the β cardiac myosin heavy-chain gene thatcreates a novel DdeI restriction-enzyme site (Geisterfer-Lowrance etal., Cell 62:999-1006 (1990)). RNA was prepared from Epstein-Barrvirus-transformed cells from both affected and unaffected member ofFamily A and sequentially amplified with primers C and D followed by C′and D′ (FIG. 1A). The amplified product was then digested with therestriction enzyme DdeI and fractionated according to size on anargarose gel. The digested samples from unaffected persons produced twofragments. The larger of these fragments was readily visible on ethidiumbromide staining and consisted of approximately 215 bp (see FIG. 1B,lanes 2 and 4). The digested samples from affected persons yielded athird visible fragment consisting of approximately 180 bp (see FIG. 1B,lanes 3 and 5), in addition to those present in unaffected persons. Thethird visible fragment is of a size which is predicted by the additionalDdeI site conferred by this mutation for FHC. Ectopic transcription ofboth the normal and the mutant allele was therefore evident in affectedpersons, as expected in an autosomal dominant disorder. Amplification ofmutant and normal sequences should occur with equal efficiencies. Theintensity of the ethidium bromide staining of the fragments, therefore,accurately reflects the relative abundance of these two transcripts anddemonstrates that the mutant and normal alleles were transcribed equallyin cell lines transformed by Epstein-Barr virus.

The detection of transcripts from both normal and mutant β cardiacmyosin heavy-chain genes in peripheral-blood cells provides a mechanismfor the rapid identification of mutations that cause FHC. The detectionof both normal and mutant genes is particularly important for FHCbecause, unlike disorders such as sickle cell anemia, different βcardiac myosin heavy-chain mutations can cause the disease in unrelatedfamilies.

Assessing the Usefulness of Detection Method for New β Cardiac MyosinHeavy-Chain Mutations in Family OO

RNA was isolated from Epstein-Barr virus-transformed cell lines derivedfrom affected persons in different, unrelated families. RNA samples wereused as the template in PCRs with nested primers (FIG. 1A, A and Bfollowed by A′ and B′), and the amplified test strands of DNA werehybridized to a β cardiac myosin heavy-chain RNA probe (FIG. 1A) forRNase protection assays (FIG. 2B). The RNase protection assay yieldedfragments which formed a complex pattern of bands, however, novelfragments were easily identified because of the homogeneity of patternsin unrelated probands (see FIG. 2B, lanes 1 through 3). A unique bandwas present in the sample analyzed in lane 3 of FIG. 2B. A second RNAsample from this person was analyzed to exclude the possibility thatthis band arose because erroneous sequences were introduced duringsequential PCRs. This analysis confirmed the presence of a new bandimplying a sequence difference between the affected person's DNA and DNAfrom an unaffected person. All other peptide-encoding regions of the βcardiac myosin heavy-chain gene was screened and no other abnormalitieswere detected in this person.

The persons family (Family QQ) was studied to determine whether thesequence difference is coinherited with disease status (part of thepedigree is shown in FIG. 3A). Ten affected family members had died ofhypertrophic cardiomyopathy before the study. Four of the deaths weresudden. Clinical evaluations of adult family members (generations I andII) identified eight affected persons (age, 28 to 68 years). Four of thefamily members were asymptomatic. All had abnormal electrocardiograms.Seven had typical left ventricular hypertrophy on two-dimensionalechocardiography, with a maximal left-ventricular-wall thickness of 1.5to 2.5 cm (mean, 2.2). Family member II-11 had apical left ventricularhypertrophy on two-dimensional echocardiography, but a maximalleft-ventricular-wall thickness of 1.0 cm and mild mitral regurgitation.The left atrial dimension was increased in all eight affected members(4.0 to 5.1 cm; mean, 4.5). None of the family members had completesystolic anterior motion of the mitral valve or evidence of a leftventricular gradient on Doppler ultrasonography. Samples from all adultswho were clinically affected on the basis of two-dimensionalechocardiography were analyzed. All of the affected family members had aband that was absent from samples derived from unaffected adult familymembers which represents a mutation in the β cardiac myosin heavy-chaingene. FIG. 2B (lines 3 through 14) shows the results of RNase protectionassays for several adults in this family. The family members aredesignated using the symbols from the family pedigree depicted in FIG.3A.

The sequence difference detected by the RNase protection assay in familymember I-1 (the arrow in FIG. 2B) also was present in all other affectedmembers of Family QQ. There was complete concordance between clinicaland molecular genetic diagnoses in all members of generations I and II.The probability of obtaining this result by chance (i.e., if themutation and disease were not linked) is 1 in 10,000 (lod score, 4.0 atθ=0).

The difference in the nucleotide base pair that accounts for the novelband in the RNase protection assays was identified by nucleic acidsequence analysis of this region of the β cardiac myosin heavy-chaingene derived from family member I-1. A guanine residue normally presentat position 832 (exon 9) was converted to an adenine residue. Thismissense mutation creates a nonconservative amino acid substitution ofglutamine for arginine (position 249), which results in a change incharge from ±1 to 0. This amino acid substitution was not previouslyidentified among 100 normal chromosomes or 50 chromosomes from unrelatedpatients with FHC. Furthermore, this arginine residue has beenstringently conserved throughout evolution and is invariant in allmuscle myosins characterized to date (Jaenicke et al., cited supra; Liewet al., Nucleic Acids Res. 18:3647-51 (1990); Dibb et al., J Mol Biol205:603-13 (1989); Jung et al. Gene 82:269-80 (1989); Karn et al., ProcNatl Acad Sci USA 80:4253-7 (1983); McNally et al., J Mol Biol210:665-71 (1989); Molina et al., J Biol Chem 262:6478-88 (1987); Rozeket al., Proc Natl Acad Sci USA 83:2128-32 (1986); Shohet et al., ProcNatl Acad Sci USA 86:7726-30 (1989);. Stedman et al., J Biol Chem265:3568-76 (1990); Strehler et al., J Mol Biol 190:291-317 (1986); Tonget al. J Biol Chem 265:2893-901 (1990); Yanagisawa et al., J Mol Biol198:143-57 (1987)).

The missense mutation abolishes an EcoRI restriction-enzyme sitenormally present in exon 9 (Jaenicke et al, cited supra and Liew et al.,cited supra) which provides an independent method of assessing geneticdiagnoses. Exon 9 sequences of the β cardiac myosin heavy-chain genewere amplified with the use of whole-blood DNA. The PCR products weredigested with EcoRI and fractionated according to size on agarose gels.The normal sequence produced two fragments that were 79 and 45 bp long.The mutated sequence lacked this EcoRI site and therefore half of thePCR product derived from affected persons was uncut (FIG. 3B, lane 1,showing results for affected member I-1, as compared with lane 2,showing results for unaffected member I-2). In each sample, two smallfragments derived from the normal allele were present. An additionallarger fragment was visible in the sample from family member I-1,confirming the loss of an EcoRI restriction-enzyme site in the mutantsequence. All adult family members were assessed by this method, and aswith RNase protection assays, there was complete agreement betweenclinical and genetic disease assignment.

Fourteen children of affected parents in this family were evaluated todetermine whether accurate diagnosis based on genetic technique ispossible without clinical evidence of FHC. None of these children, whoranged in age from 1 to 20 years, were previously known to be affectedand none had symptoms suggestive of FHC. Their two-dimensionalechocardiographic and electrocardiographic findings are shown in Table Ibelow. TABLE 1 Results of Clinical and Genetic Analysis of GenerationIII of Family QQ. Family Age 2-Dimensional Geno- Member (Yr)Echocardiogram Electrocardiogram Type III-1 20 Normal Abnormal Q wave,inferior + T-wave inversion III-2 19 Septal and free- Left ventricular +wall hypertrophy hypertrophy inferior T-wave inversion III-3 16 NormalNormal − III-4 14 Normal Left ventricular + hypertrophy inferior T-waveinversion III-5 12 Normal Normal − III-6 7 Normal Inferior T-waveinversion + III-7 14 Normal Normal − III-8 11 Normal Normal − III-9 2Normal Normal + III-10 8 Normal QRS complex prolonged + for age III-11 6Normal Normal − III-12 4 Normal Normal + III-13 3 Normal Normal − III-141 Normal Normal −Only one child (III-2) had findings diagnostic of FHC. Two children(III-1 and III-4) had subtle features of focal hypertrophy noted by oneinvestigator but a definite clinical diagnosis for FHC could not bemade. Five children had electrocardiographic abnormalities, includingleft ventricular hypertrophy (III-2 and III-4), abnormal Q wave (III-1),T-wave abnormalities (III-1, III-2, III-4, and III-6) and a QRS complexthat was longer than expected for age (III-10) (Perry et al., J.Pediatrics 97:677-87 (1980)). A genetic diagnosis, based on a 5-ml bloodsample, was made in all 14 children without knowledge of the clinicalfindings. DNA digestion with the restriction enzyme EcoRI (FIG. 3B,lanes 3-15) and RNase protection assays were performed on PCR productsamplified from exon 9. Each analysis identified seven children with amissense mutation of the β cardiac myosin heavy-chain gene at amino acidresidue 249, and the results were completely concordant. These sevenchildren included all children with any abnormalities present ontwo-dimensional echocardiograms or electrocardiograms. A geneticdiagnosis of FHC was also made in two children (III-9 and III-12; ages 2and 4 years) who had completely normal clinical-studies.Clinical Evaluation

Blood samples were obtained from the respective family members.Clinical, electrocardiographic, and echocardiographic assessments wereperformed as previously described (Jarcho et al., cited supra). Thediagnosis of hypertrophic cardiomyopathy was based on he demonstrationof unexplained hypertrophy of either ventricle or both ventricles.Clinical records, family histories, or both were obtained to determinethe number of disease-related deaths, the number of sudden deaths(disease-related deaths due to unexpected cardiac arrest or abruptcirculatory collapse), and the age at death or the current age of allaffected members of each family. Kaplan-Meier product-limit survivalcurves were produced as described elsewhere (Kaplan et al., J. Am. Stat.Assoc. 53:457-81 (1958); Lee, “Statistical Methods for Survival DataAnalysis”, Belmont, Calif., Lifetime Learning Publications, (1980)).These curves were compared according to the log-rank method of Peto andPeto (Cox et al., “Analysis of Survival Data”, London, Chapman and Hall,(1984)). All P values were calculated with the assumption of atwo-tailed distribution.

Strategy for the Detection of Mutations

The polymerase chain reaction (PCR) was used to amplify the sequences ofβ cardiac myosin heavy-chain genes derived from RNA isolated fromnucleated blood cells obtained from affected family members that weretransformed by the Epstein-Barr virus (FIG. 4A). Both normal andmutationally altered β cardiac myosin heavy-chain RNA were detected inthese cell lines as described above. Amplified sequences were hybridizedto RNA probes derived from an unaffected member, and an RNase Aprotection assay was performed as described above. Both sense andantisense riboprobes were used to increase the probability ofidentifying all mutations. The entire β cardiac myosin heavy-chaincoding sequence of each proband was examined to determine whether any ofthese sequences contained more than one mutation. Amplified DNA samplesyielding abnormal RNase cleavage patterns were reanalyzed with new DNAisolates to exclude artifacts arising from the PCR and were thensubjected to nucleotide-sequence analysis. Throughout this example,mutations are denoted by the three-letter code for the normal aminoacid, followed by the residue number and the code for the predictedamino acid sequence resulting from each mutation.

Templates for the Riboprobes

Twenty-five base oligonucleotide primers (containing nucleotidesequences as numbered by Jaenicke et al., cited supra and a selectedrestriction-enzyme site) were used to reverse-transcribe and amplifyseven segments of normal human β cardiac myosin heavy-chain RNA (FIG.4A). One segment (3421 through 3811) could not be amplified from RNAwith PCR and was produced by amplifying exon 27 from human DNA. Eachamplified product was cloned into a Blue-script SK vector (Stratagene)according to standard procedures (Ausubel et al., Current Protocols inMolecular Biology, New York, Green Publishing, 1989 (1991 update)). Theeight different β cardiac myosin heavy-chain clones were linearized byrestriction-enzyme digestion and transcribed with the use of T3 or T7RNA polymerase.

DNA for Screening

Segments of β cardiac myosin heavy-chain cDNA were obtained by nestedPCR amplification of cDNA reverse-transcribed from peripheral-leukocyteRNA as described above, or individual exons were amplified from genomicDNA. cDNA segments, numbered 1 through 6 (FIG. 4A) were amplified withthe former approach. The diluted product from an initial PCRamplification with outer primers was used as the template for a secondround of PCR amplification with an inner-primer pair. This techniquecould not be used throughout the example because of difficulties inamplifying some RNA sequences encoding the rod region. These areas werescreened by amplifying the sequences of individual exons. Section 1203through 2398 (corresponding to exons 13 through 20) was screened withDNA templates produced according to both techniques, with identicalfindings.

The following nucleotide numbers are those of the primers used in thenested PCR amplification of cDNA (all were 25-mers, each numbered by its5′ residue according to the cDNA sequence (Jaenicke et al., citedsupra); see FIG. 4A; 1, outer 20 to 475, inner 50 to 450; 2, outer 401to 1235, inner 425 to 800 and 750 to 1175; 3, outer 750 to 1700, inner1101 to 1600; 4, outer 1501 to 2450, inner 1526 to 2025 and 1925 to2424; 5, outer 2300 to 3301, inner 2325 to 2825 and 2726 to 3276; and 6,outer 4401 to 5105, inner 4449 to 5080. The following are the primersfor the exons (all 25-mers, each numbered by its 5′ residue according tothe gene sequence (Jaenicke et al., cited supra)): 27, 19178 to 19597;28, 19740 to 20048; 29, 20101 to 20279; 30, 20973 to 21257; 31, 21689 to21954; 32, 22033 to 22313; 35, 23567 to 23848; 36, 23902 to 24088; 37,24123 to 24457; 38, 25293 to 25470; 39, 25508 to 25698; and 40, 26539 to26724.

Linkage Analyses

Sequence variants identified in a proband were used to assess geneticlinkage between the disease status of family members and the β cardiacmyosin heavy-chain gene. Lod (logarithm of the odds) scores werecalculated with the LINKMAP program (Lathrop et al., PNAS USA 81:3443-6(1984), for a recombination fraction (θ) of 0.0, with a penetrance of0.95 and an allele frequency of the sequence variant of 0.05. Lod scoresof families with the same mutation were combined. A lod score greaterthan 1.3 indicates that the odds in support of linkage are higher than20 to 1.

Study of Twenty-Five Kindreds with Familial Hypertrophic Cardiomyopathy

EXAMPLE 2 Determination of the Proportion of Families with HypertrophicCardiomyopathy Caused by Myosin Heavy-Chain Mutations

Twenty-five families were studied whose members have hypertrophiccardiomyopathy. Preliminary research had indicated that major structuralabnormalities of the α or β cardiac myosin heavy-chain genes are not acommon cause of FHC. RNase protection assays therefore were used toscreen directly for point mutations or other small alterations in the βcardiac myosin heavy-chain gene which encodes the predominant isoform ofmyosin expressed in the ventricles of adults (Mahdavi et al., Nature297:659-64 (1982); Lomprei et al., J. Biol. Chem. 259:6437-46 (1987)).The following general methodology was used in the example below.

The affected members of these twenty-five families have features typicalof hypertrophic cardiomyopathy as assessed by physical examination,two-dimensional Doppler echocardiography and electrocardiography. Thedisease was inherited as an autosomal dominant trait in all cases, asdocumented by the history or clinical evaluation (or both) of relatives.The families were of European descent and were unrelated. In three ofthe families (A, B, and QQ), the disease locus was known or believed tobe linked to chromosome 14 band q1 from Example 1 above andGeisterfer-Lowrance et al. (Cell 61:999-1006 (1990)). The chromosomallinkage of the disease locus in all of the other families was unknown. Amutation had been previously identified in Families A and B from alimited analysis of the β cardiac myosin heavy-chain gene(Geisterfer-Lowrance et al., cited supra and Example 1 above. Oneproband was selected from each family for genetic analysis. A proband isderived from an affected member of a family who is selected as therepresentative subject for study. The entire coding sequence of the βcardiac myosin heavy-chain gene was screened to identify mutations inthese probands using RNase protection assays. These assays identifiednine different variants from the normal sequence of the β cardiac myosinheavy-chain gene. These nine variants were found in fourteen of theprobands and two of these variants are shown in FIG. 4B.

The nine variants were characterized by nucleotide-sequence analysis.All the variants corresponded to single-nucleotide substitutions. Eightof the variants were transitions (G to A, or C to T) and one was atransversion (G to C). Six of the eight transitions occurred at a CGdinucleotide which is a common site of mutations in human disease loci(Youssoufian et al., Am. J. Hum. Genet. 42:718-25 (1988); Green et al.,Nucleic Acids Res 18:3227-31 (1990); Rideout et al., Science 249:1288-90(1990)). Seven DNA variants changed the coding sense of the β cardiacmyosin heavy-chain gene (FIG. 5) and two did not alter the encoded aminoacid sequence. These two variants were silent polymorphisms, both ofwhich were found in unaffected family members. All seven variants thatchanged the coding sense affected residues in the amino-terminal half ofthe β cardiac myosin heavy-chain polypeptide (FIG. 5). Four variantsequences were found in two or more probands. These included themutation of arginine to glutamine at residue 403 (Arg403Gln), which wasinitially detected in affected members of Family A (Geisterfer-Lowranceet al., cited supra) and is also found in the proband from Family SS.

There are three findings that support the position that the sevensequence variants were mutations causing FHC. First, there was completeconcordance between genotype and disease status in all adult relativesof each proband in whom a mutation was identified. Linkage analysesprovided statistically significant information about six of the sevenmutations since many affected families were large (see Table II below).TABLE 2 β Cardiac Myosin Heavy-Chain Gene Mutations and AssociatedLaboratory and Clinical Features in Families with Familial HypertrophicCardiomyopathy. Gene Mutation Arg249 Arg403 Arg453 Arg453 Gly584 Val606Glu924 Glu949 Gln Gln Cys Cys + HYBRID Arg Met Lys Lys FeatureNucleotide change* G832A G1294A C1443T C1443T G1836C G1902A G2856AG2931A Change in charge† −1 −1 −1 −1 +1 0 +2 +2 Families affected QQ A,SS E B LL, DD L, BB, G H YY Lod score 4.0 15.9 3.9 4.4 1.4 3.5 1.1 2.2No. of members affected 24 44 13 13 5 18 2 2 Mortality No. ofdisease-related deaths 10 21 9 4 2 1 0 0 No. of sudden deaths 4 9 6 2 21 0 0 Average age at death (yr)‡ 49 ± 22 33 ± 15 30 ± 12 35 ± 17 19 ± 613 — —*These mutations are designated by the normal residue and its position(numbered as described by Jaenicke et al.) followed by the mutantresidue.†Values are changes in the net charge of the polypeptide, based oncharges of the amino acid at pH7.‡The average age at death was calculated for all deaths related tofamilial hypertrophic cariomyopathy.Plus-minus values are means ± SD.Differences in lod scores reflected only differences in family sizebecause these analyses were fully informative for all members. Second,each sequence variant predicted that the encoded amino acid residuewould be altered and each altered amino acid was one that has beenentirely conserved during the evolution of a vertebrate striated muscleimplying functional importance. Third, these variants were not found inanalyses of more than 180 normal chromosomes.

Previous studies of Family B demonstrated that affected members had anα/β cardiac myosin heavy-chain hybrid gene in addition to nonrearrangedα and β myosin heavy-chain genes (Tanigawa et al., Cell 62:991-8(1990)). The proband from this family was included in theabove-described analyses and the Arg453Cys mutation was identified in anonrearranged β cardiac myosin heavy-chain gene. This mutation was alsoidentified in affected members of an unrelated family, Family E, all ofwhom lacked the hybrid gene. The natural history of the disease inaffected members of these two families appeared to be similar. It wasdetermined that the missense mutation and not the hybrid gene wasresponsible for the FHC in both families. Because the Arg453Cys mutationoccurred in affected members of two unrelated families who had a similarphenotype,

Comparison of the Spectrum of Clinical Features in Affected Members ofFamilies With Particular Mutations of the β Cardiac Myosin Heavy-ChainGene

The spectrum of clinical features of FHC was compared in affectedmembers of families in which a mutation of the β cardiac myosinheavy-chain gene was identified. The incidence of angina, dyspnea, andsyncope among members of a family with a given mutation wasindistinguishable from the incidence among members of families withdifferent mutations. The severity of ventricular hypertrophy as assessedby two-dimensional echocardiography also was indistinguishable amongfamilies with different mutations. The range of values for the maximalthickness of the left ventricular wall in patients with the samemutation was not significantly different from that in affected memberswith different mutations.

The Determination of Whether the Presence of a Particular β CardiacMyosin Heavy-Chain Gene Mutation is Prognostic

Several indexes of survival in relation to genotype (Table 2 in FIG. 6)combining data on families with identical mutations were compared todetermine whether the presence of a particular β cardiac myosinheavy-chain gene mutation is prognostic. Disease-related deaths wereless frequent in families with the Val606Met mutation than in familieswith the Arg249Gln, Arg403Gln, or Arg453Cys mutation (Table 2). Patientswith the Arg249Gln mutation had a significantly longer life expectancy(Table 2) than those with the Arg403Gln mutation (P−0.027) or those withthe Arg453Cys mutation (P=0.023). Survival analysis of the smallfamilies with the Gly584Arg, Glu924Lys, and Glu949Lys mutations providedlittle information.

Sufficient numbers of affected members were available for Kaplan-Meierproduct-limit survival curves to be produced for five mutations. Thedata for Family B and Family E were combined because the survival curvefor patients with the Arg453Cys mutation involving the hybrid gene(Family B) was indistinguishable from the curve for the patients withoutthis gene (Family E). These analyses confirmed that the Val606Metmutation was associated with longer survival than was the Arg453Cysmutation (P=0.002) or the Arg403Gln mutation (P=0.002). The Arg249Glnmutation appeared to produce an intermediate phenotype. Survival waslonger among patients with this mutation than those with the Arg453Cysmutation (P=0.027) or those with the Arg403Gln mutation (P=0.015), buttended to be shorter than survival among patients with the Val606Metmutation (P=0.067). Survival among patients with the Arg453Cys mutation(with or without the hybrid gene) was similar to survival among thosewith the Arg403Gln mutation (P=0.79). Both mutations were associatedwith a particularly poor prognosis.

Results of Study

Mutations in the β cardiac myosin heavy-chain gene was identified in 12of 25 families with FHC as shown in Table 2. Seven different missensemutations were found that are located in the head or head-rod junctionregion of the myosin heavy chain. No mutations were detected in he rodregion. Six of the seven nucleotide substitutions altered the charge ofthe encoded amino acids and were particularly likely to lead to regionalconformational changes in the polypeptide. The survival of affectedfamily members, but not the extent of cardiac hypertrophy or symptoms,appears to be influenced by the particular mutation.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for detecting the presence or absence of a mutationassociated with hypertrophic cardiomyopathy for facilitating thediagnosis of hypertrophic cardiomyopathy, comprising: amplifying βcardiac myosin heavy-chain DNA forming an amplified product; anddetecting the presence or absence of a mutation associated withhypertrophic cardiomyopathy in the amplified product therebyfacilitating the diagnosis of hypertrophic cardiomyopathy.
 2. The methodof claim 1 wherein the hypertrophic cardiomyopathy is familialhypertrophic cardiomyopathy, or sporadic hypertrophic cardiomyopathy. 3.The method of claim 2 wherein the mutation associated with hypertrophiccardiomyopathy is a point mutation or a missense mutation.
 4. The methodof claim 1 wherein the mutation associated with hypertrophiccardiomyopathy is of a size less than the amplified product.
 5. Themethod of claim 1 wherein the β cardiac myosin heavy-chain DNA is cDNAreverse transcribed from RNA.
 6. The method of claim 5 wherein the RNAis obtained from nucleated blood cells.
 7. The method of claim 1 whereinthe presence or absence of the mutation associated with hypertrophiccardiomyopathy is detected by combining the amplified product with anRNA probe completely hybridizable to normal β cardiac myosin heavy-chainDNA forming a hybrid double strand having an RNA and DNA strand, thehybrid double strand having an unhybridized portion of the RNA strand atany portion corresponding to a hypertrophic cardiomyopathy associatedmutation in the DNA strand; and detecting the presence or absence of anunhybridized portion of the RNA strand as an indication of the presenceor absence of a hypertrophic cardiomyopathy associated mutation in thecorresponding portion of the DNA strand.
 8. The method of claim 2wherein the presence or absence of the mutation associated with familialhypertrophic cardiomyopathy is detected by combining the amplifiedproduct with an RNA probe completely hybridizable to normal β cardiacmyosin heavy-chain DNA forming a hybrid double strand having an RNA andDNA strand, the hybrid double strand having an unhybridizedribonucleotide of the RNA strand at any portion corresponding to afamilial hypertrophic cardiomyopathy associated point mutation in theDNA strand; contacting the hybrid double strand with an agent capable ofdigesting an unhybridized portion of the RNA strand; and detecting thepresence or absence of an unhybridized ribonucleotide of the RNA strandas an indication of the presence or absence of a familial hypertrophiccardiomyopathy associated point mutation in the correspondingdeoxyribonucleotide of the DNA strand.
 9. The method of claim 1 whereinthe β cardiac myosin heavy-chain DNA is amplified using a polymerasechain reaction.
 10. The method of claim 9 wherein the polymerase chainreaction is performed with nested primers.
 11. The method of claim 1wherein said hypertrophic cardiomyopathy-associated mutations areselected from the group consisting of G832A; C1443T; G1836C; G1902A;G2856A; and G2931A.
 12. A method according to claim 1 further comprisingdetecting the presence of more than one target sequence in said DNA. 13.A method according to claim 12 wherein said more than one targetsequence is a hypertrophic cardiomyopathy-associated mutation selectedfrom the group consisting of G832A; G1294A; C1443T; G1836C; G1902A;G2856A; and G2931A.
 14. A method of claim 1, wherein the β cardiacmyosin heavy-chain RNA is obtained from a from said sample a cell samplefrom a subject being tested for hypertrophic cardiomyopathy; anddiagnosing the subject for hypertrophic cardiomyopathy by detecting thepresence or absence of a familial hypertrophic cardiomyopathy-associatedmutation in the RNA as an indication of hypertrophic cardiomyopathy. 15.A method of claim 14, wherein the method for diagnosing hypertrophiccardiomyopathy is non-invasive.
 16. A set of DNA oligonucleotide primersfor amplifying β-cardiac myosin heavy-chain DNA comprising, at least twooligonucleotides which amplify β-cardiac myosin heavy-chain DNA, saidset of oligonucleotide primers being useful for facilitating thediagnosis of hypertrophic cardiomyopathy by being capable of detecting ahypertrophic cardiomyopathy-associated mutation.
 17. The set of primersof claim 16 having at least four oligonucleotides.
 18. Theoligonucleotide primers for amplifying β-cardiac myosin heavy-chain DNAof claim 16, said primers comprising at least two oligonucleotideswherein each of the oligonucleotides is selected from the groupconsisting of: (SEQ ID NO:1) 5′ CAAGGATCGCTACGGCTCCTGGAT 3′, (SEQ IDNO:2) 5′ GCGGATCCAGGTAGGCAGACTTGTCAGCCT 3′, (SEQ ID NO:3) 5′ATGCCAACCCTGCTCTGGAGGCCT 3′, (SEQ ID NO:4) 5′ CTTCATGTTTCCAAAGTGCATGAT3′, (SEQ ID NO:5) 5′ CTGGGCTTCACTTCAGAGGAGAAAA 3′, (SEQ ID NO:6) 5′GCGGTACCCCAGCAGCCCGGCCTTGAAGAA 3′, (SEQ ID NO:7) 5′GGGAATTCGCGGAGCCAGACGGCACTGAAG 3′, (SEQ ID NO:8) 5′CCCTCCTTCTTGTACTCCTCCTGCTC 3′, (SEQ ID NO:9) 5′CAACTCATCACCACTCTCTTCCATC 3′, and (SEQ ID NO:10) 5′GCTGAGCCTAGCAGATTCATGGCAC 3′.


19. A kit useful for facilitating the diagnosis of hypertrophiccardiomyopathy, comprising: a first container holding an RNA probecompletely hybridizable to the β cardiac myosin heavy chain DNA, whereinsaid RNA probe is capable of detecting a hypertrophiccardiomyopathy-associated mutation; a second container holding primersuseful for amplifying β cardiac myosin heavy-chain DNA; and instructionsfor using the components of the kit to detect the presence or absence ofa hypertrophic cardiomyopathy-associated mutation in amplified β cardiacmyosin heavy-chain DNA for facilitating the diagnosis of hypertrophiccardiomyopathy.
 20. A kit of claim 19 further comprising a thirdcontainer holding an agent for digesting unhybridized RNA.